Transfer of antigen from human B cells to dendritic cells

The cooperation of B lymphocytes with other antigen presenting cells (APCs) is often necessary in the efficient processing and presentation of antigen. Herein, we describe a mechanism by which B cells physically interact with dendritic cells (DCs) resulting in the transfer of B cell receptor (BCR)-enriched antigen to these APCs. Antigen transfer involves direct contact between the two cells followed by the capture of B cell derived membrane and intracellular components. Strikingly, DCs acquire greater amounts of antigen by transfer from B cells than by endocytosis of free antigen. Blocking scavenger receptor A, a DC surface receptor involved in membrane acquisition, abrogates these events. We propose that antigen transfer from B cells to DCs results in a more focused immunologic response due to the selective editing of Ag by the BCR.     

Stability of the B cell antigen receptor modulates its signaling and antigen-targeting functions

The binding of antigens to the B cell antigen receptor (BCR) results in the initiation of signaling cascades and the internalization of the antigens for processing and presentation. Recent studies indicate that antigen binding destabilizes the BCR as a mechanism to down-regulate B cell responses. Two point mutations in the transmembrane domain of murine membrane IgM (mIgM) (YS to VV) weaken the interaction of mIgM with Igalpha/Igbeta heterodimer, resulting in a destabilized BCR. Using muYS/VV BCR, effects of the destabilized BCR on the functions of an endogenous wild-type mIgG(2a) BCR were analyzed. The muYS/VV BCR is defective in signaling and does not target antigens to late endocytic compartments for processing and presentation. Coligation of the muYS/VV BCR with the endogenous BCR interferes with antigen-targeting functions of the endogenous BCR. Thus, the destabilized BCR has a dominant effect, down-regulating the function of stable wild-type BCR. The ability of the destabilized BCR to influence the stable BCR may play an important role in turning off B cell responses for antigen-driven anergy and tolerance.     

The effect of B cell receptor signaling on antigen endocytosis and processing

B cell receptor (BCR)-mediated antigen processing and presentation involves both the BCR-mediated internalization and processing of cognate antigen as well as the formation and expression of antigenic peptide-MHC class II complexes. While BCR signaling is known to result in changes in the biosynthesis and intracellular trafficking of class II molecules, the effect of BCR signaling on the cell biology of antigen endocytosis and processing is less clear. Therefore, the effect of BCR signaling on the cell biology of fluid phase antigen endocytosis, processing and presentation was analyzed in both B cell lines or in normal splenic B cells. The results demonstrate that BCR signaling alters neither the global level of fluid phase antigen endocytosis nor the duration of intracellular persistence of fluid phase internalized antigen. Moreover, while BCR signal does result in an increase in the level of total cell surface MHC class II molecules as well as specific peptide-class II complexes, stimulation failed to alter the fraction of class II molecules loaded with antigen-derived peptide. These results indicate that while BCR-mediated signaling elicits an increase in the expression of antigenic peptide-class II complexes, signaling does not augment antigen presentation by profoundly altering the basic biology of antigen endocytosis and processing. These results also demonstrate that the high efficiency of BCR-mediated antigen processing (when compared to fluid phase antigen processing) is likely to occur independent of BCR signaling-induced global alterations in the biology of endocytosis, processing and presentation. This finding suggests that if BCR signaling augments the efficiency of processing of cognate antigen, it must impact unique aspects of BCR-mediated antigen processing, such as the intracellular persistence of internalized antigen-BCR complexes.     

The Effect of B Cell Receptor Signaling on Antigen Endocytosis and Processing

B cell receptor (BCR)-mediated antigen processing and presentation involves both the BCR-mediated internalization and processing of cognate antigen as well as the formation and expression of antigenic peptide-MHC class II complexes. While BCR signaling is known to result in changes in the biosynthesis and intracellular trafficking of class II molecules, the effect of BCR signaling on the cell biology of antigen endocytosis and processing is less clear. Therefore, the effect of BCR signaling on the cell biology of fluid phase antigen endocytosis, processing and presentation was analyzed in both B cell lines or in normal splenic B cells. The results demonstrate that BCR signaling alters neither the global level of fluid phase antigen endocytosis nor the duration of intracellular persistence of fluid phase internalized antigen. Moreover, while BCR signal does result in an increase in the level of total cell surface MHC class II molecules as well as specific peptide-class II complexes, stimulation failed to alter the fraction of class II molecules loaded with antigen-derived peptide. These results indicate that while BCR-mediated signaling elicits an increase in the expression of antigenic peptide-class II complexes, signaling does not augment antigen presentation by profoundly altering the basic biology of antigen endocytosis and processing. These results also demonstrate that the high efficiency of BCR-mediated antigen processing (when compared to fluid phase antigen processing) is likely to occur independent of BCR signaling-induced global alterations in the biology of endocytosis, processing and presentation. This finding suggests that if BCR signaling augments the efficiency of processing of cognate antigen, it must impact unique aspects of BCR-mediated antigen processing, such as the intracellular persistence of internalized antigen-BCR complexes.     

LFA-1/ICAM-1 interaction lowers the threshold of B cell activation by facilitating B cell adhesion and synapse formation

The integrin LFA-1 and its ligand ICAM-1 mediate B cell adhesion, but their role in membrane-bound antigen recognition is still unknown. Here, using planar lipid bilayers and cells expressing ICAM-1 fused to green fluorescence protein, we found that the engagement of B cell receptor (BCR) promotes B cell adhesion by an LFA-1-mediated mechanism. LFA-1 is recruited to form a mature B cell synapse segregating into a ring around the BCR. This distribution is maintained over a wide range of BCR/antigen affinities (10(6) M(-1) to 10(11) M(-1)). Furthermore, the LFA-1 binding to ICAM-1 reduces the level of antigen required to form the synapse and trigger a B cell. Thus, LFA-1/ICAM-1 interaction lowers the threshold for B cell activation by promoting B cell adhesion and synapse formation.     

A role for MHC class II antigen processing in B cell development

For mature B cells, the encounter with foreign antigen results in the selective expansion of the cells and their differentiation into antibody secreting cells or memory B cells. The response of mature B cells to antigen requires not only antigen binding to and signaling through the B cell antigen receptor (BCR) but also the processing and presentation of the BCR bound antigen to helper T cells. Thus, in mature B cells, the ability to process and present antigen to helper T cells plays a critical role in determining the outcome of antigen encounter. In immature B cells, the binding of antigen results in negative selection of the B cell, inducing apoptosis, anergy or receptor editing. Negative selection of immature B cells requires antigen induced signaling through the BCR, analogous to the signaling function of the BCR in mature B cells. However, the role of class II antigen processing and presentation in immature B cells is less well understood. Current evidence indicates that the ability to process and present antigen bound to the BCR is a late acquisition of developing B cells, suggesting that during negative selection B cells may not present BCR bound antigen and interact with helper T cells. However, the expression of class II molecules is an early acquisition of B cells and recent evidence indicates that the expression of class II molecules early in development is required for the generation of long lived mature B cells. Here we review our current understanding of the processing and presentation of antigen by mature B cells and the role for antigen processing and class II expression during B cell development.     

A Role for MHC Class II Antigen Processing in B Cell Development

For mature B cells, the encounter with foreign antigen results in the selective expansion of the cells and their differentiation into antibody secreting cells or memory B cells. The response of mature B cells to antigen requires not only antigen binding to and signaling through the B cell antigen receptor (BCR) but also the processing and presentation of the BCR bound antigen to helper T cells. Thus, in mature B cells, the ability to process and present antigen to helper T cells plays a critical role in determining the outcome of antigen encounter. In immature B cells, the binding of antigen results in negative selection of the B cell, inducing apoptosis, anergy or receptor editing. Negative selection of immature B cells requires antigen induced signaling through the BCR, analogous to the signaling function of the BCR in mature B cells. However, the role of class II antigen processing and presentation in immature B cells is less well understood. Current evidence indicates that the ability to process and present antigen bound to the BCR is a late acquisition of developing B cells, suggesting that during negative selection B cells may not present BCR bound antigen and interact with helper T cells However, the expression of class II molecules is an early acquisition of B cells and recent evidence indicates that the expression of class II molecules early in development is required for the generation of long lived mature B cells. Here we review our current understanding of the processing and presentation of antigen by mature B cells and the role for antigen processing and class II expression during B cell development.     

Lowering the affinity between antigen and the B cell receptor can enhance antigen presentation

The B cell receptor (BCR) enables antigen-specific B cells to bind, internalize and target antigens for processing into small peptide fragments. These epitopes are then expressed on the plasma membrane in association with MHC class II molecules for recognition by CD4+ T cells. The affinity of the interaction between the BCR and antigen plays an important part in determining T cell epitope generation. In this report we provide evidence that the efficiency of antigen presentation by specific B cells does not need to be directly proportional to antigen/BCR affinity. We show that increased presentation can result from lowering the affinity of the antigen/BCR interaction. This finding suggests a novel mechanism by which B cells can recruit T cell help and obtain survival signals. Activation of these cells may have consequences for the generation of the B cell repertoire.     

Lack of induced co-stimulation as a result of complement receptor 2 (CR2) ligation on mouse splenic B cells

B cells act as efficient antigen-presenting cells if they acquire antigen via membrane-bound Ig [termed the B cell receptor (BCR)]. Ligation of the BCR leads to antigen internalization, processing and presentation to CD4+ T cells in association with MHC class II molecules. Ligation of the BCR also leads to the generation of activation signals. One short-term consequence of this is the up-regulation of co-stimulatory molecule expression by the B cell, allowing full T cell activation. Other antigen receptors expressed by B cells can also mediate efficient antigen presentation to CD4+ T cells. Ligating one such receptor, complement receptor 2 (CR2), has also been described to induce co-stimulatory molecule expression. If correct, this may have serious consequences for ensuring the specificity of the resultant B cell response. We have therefore investigated the effects of ligating both the BCR and CR2 independently of each other, as well as with reagents to cross-link the two receptors, in order to clarify these findings. In contrast to the effects seen upon BCR ligation, we find no evidence for co-stimulatory molecule up-regulation following CR2 ligation. As antigen presentation in the absence of co-stimulation may lead to the induction of tolerogenic or regulatory signals being delivered to T cell populations, these findings imply that the role of CR2 in B cell-mediated antigen presentation is different from that of the BCR.     

Simvastatin inhibits the MHC class II pathway of antigen presentation by impairing Ras superfamily GTPases

Statins are widely used hypocholesterolemic drugs that inhibit 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, a rate-limiting enzyme of the mevalonate pathway whose biosynthetic endproduct is cholesterol. As a result of this activity, statins may perturb the composition of cell membranes, resulting in lipid raft disruption. Furthermore, by inhibiting protein prenylation, a process also dependent on mevalonate, statins block membrane targeting and activity of small GTPases. Antigen uptake, processing and presentation involve the interplay of Rab and Rho family GTPases. Furthermore, lipid rafts have been implicated both in antigen internalization by the BCR and in MHC class II clustering at the immunological synapse. Here we have addressed the effects of simvastatin on antigen processing and presentation by human B cells and dendritic cells. The results show that simvastatin potently suppresses tetanus toxoid processing and presentation to CD4+ T cells by HLA-DR by inhibiting protein antigen uptake through both receptor-mediated endocytosis and macropinocytosis. This effect can be largely accounted for by defective prenylation of Rho and Rab GTPases in the absence of any measurable perturbation of lipid rafts. In addition, simvastatin was found to preferentially affect the invariant chain-dependent MHC class II pathway, thereby identifying this route of antigen processing and presentation as a selective target of statins.     

Lipid rafts unite signaling cascades with clathrin to regulate BCR internalization

A major function of the B cell is the internalization of antigen through the BCR for processing and presentation to T cells. While there is evidence suggesting that lipid raft signaling may regulate internalization, the molecular machinery coordinating these two processes remains to be defined. Here we present a link between the B cell signaling and internalization machinery and show that Src-family kinase activity is required for inducible clathrin heavy chain phosphorylation, BCR colocalization with clathrin, and regulated internalization. An analysis of different B cell lines shows that BCR uptake occurs only when clathrin is associated with rafts and is tyrosine phosphorylated following BCR crosslinking. We therefore propose that lipid rafts spatially organize signaling cascades with clathrin to regulate BCR internalization.     

Memory B cells from a subset of treatment‐naïve relapsing‐remitting multiple sclerosis patients elicit CD4+ T‐cell proliferation and IFN‐γ production in response to myelin basic protein and myelin oligodendrocyte glycoprotein

Recent evidence suggests that B‐ and T‐cell interactions may be paramount in relapsing‐remitting MS (RRMS) disease pathogenesis. We hypothesized that memory B‐cell pools from RRMS patients may specifically harbor a subset of potent neuro‐APC that support neuro‐Ag reactive T‐cell proliferation and cytokine secretion. To test this hypothesis, we compared CD80 and HLA‐DR expression, IL‐10 and lymphotoxin‐α secretion, neuro‐Ag binding capacity, and neuro‐Ag presentation by memory B cells from RRMS patients to naïve B cells from RRMS patients and to memory and naïve B cells from healthy donors (HD). We identified memory B cells from some RRMS patients that elicited CD4⁺ T‐cell proliferation and IFN‐γ secretion in response to myelin basic protein and myelin oligodendrocyte glycoprotein. Notwithstanding the fact that the phenotypic parameters that promote efficient Ag presentation were observed to be similar between RRMS and HD memory B cells, a corresponding capability to elicit CD4⁺ T‐cell proliferation in response to myelin basic protein and myelin oligodendrocyte glycoprotein was not observed in HD memory B cells. Our results demonstrate for the first time that the memory B‐cell pool in RRMS harbors neuro‐Ag specific B cells that can activate T cells.     

Influence of B cell receptor ligation and TCR affinity on T-B collaboration in vitro

Co-culture of purified T and B cells obtained from cytochrome c-specific TCR- and hen egg lysozyme (HEL)-specific Ig-transgenic mice was used to examine the role of B cell receptor (BCR) ligation and TCR affinity on the efficiency of T-B cell collaboration. The results showed that BCR ligation of naive B cells with HEL was not required for effective presentation of high-affinity antigen to T cells, although it did enhance activation and division of both T and B cells. Anergic B cells were also effective at presentation of high-affinity antigen and proliferated more than naive B cells in response to T cell help, due to prior exposure to antigen in vivo. Despite the fact that induction of CD86 on anergic B cells following BCR ligation was suboptimal, these cells supported T cell activation and survival in culture as efficiently as naive B cells exposed to HEL. In contrast, when the low-affinity antigen mls-3^a served as the T cell stimulus, BCR ligation was essential to elicit a detectable T cell response. Thus the in vitro model demonstrates that co-stimulation is not an absolute requirement for effective antigen presentation and delivery of T cell help to B cells. Rather, the cooperative effects of BCR ligation and TCR affinity determine the relative requirement for co-stimulation.     

Characterization of MHC-II antigen presentation by B cells and monocytes from older individuals

In this study we examine the effects of aging on antigen presentation of B cells and monocytes. We compared the antigen presentation function of peripheral blood B cells from young and old subjects using a system that specifically measures the B cell receptor (BCR)-mediated MHC-II antigen presentation. Monocytes were studied as well. Overall the mean magnitude of antigen presentation of soluble antigen and peptide was not different in older and younger subjects for both B cells and monocytes. Older subjects, however, showed increased heterogeneity of BCR-mediated antigen presentation by their B cells. The magnitude and variability of peptide presentation, which do not require uptake and processing, were the same between groups. Presentation by monocytes had similar variability between the older and younger subjects. These data suggest that poor B cell antigen processing, which results in diminished presentation in some older individuals may contribute to poor vaccine responses.     

Contrasting cytoskeletal regulation of MHC class II peptide presentation by human B cells or dendritic cells

MHC class II-mediated antigen presentation by B lymphocytes or dendritic cells (DC) initiates CD4+ T lymphocyte activation. In B lymphocytes, MHC class II peptide presentation has been characterised by recruitment of MHC class II, F-actin and lipid rafts to the B cell-T cell immunological synapse. We now show that MHC class II engagement in B lymphocytes induced lipid raft-independent Rho and Rac activation and that inhibition of either Rho-GTPase activation or actin polymerisation in the B cell abrogated T cell activation without altering B cell-T cell conjugate formation. Short-hairpin RNA studies excluded a role for the Cdc42 effector Wiskott-Aldrich syndrome protein. In contrast, antigen presentation by DC was Rho-GTPase-independent although actin was recruited to the DC-T cell interaction site. Moreover, actin depolymerisation in the DC significantly increased T cell activation without altering the number of DC-T cell conjugates. Finally we show that stable recruitment of HLA-DR to the site of the immunological synapse is not a uniform observation in DC and demonstrate reduced HLA-DR expression at the site of microtubule organising centre polarization. Therefore although actin accumulates in DC and B lymphocytes at the immunological synapse with antigen-specific T lymphocytes, this does not reflect comparable functional roles of their actin cytoskeletons in antigen presentation.     

Deficient antigen processing of a protein quaternary structure can be overcome by receptor-mediated uptake

Human chorionic gonadotropin (hCG) is a dimer of non-covalently associated alpha (hCG-α) and beta (hCG-β) subunits. This molecule was used to study whether receptor-mediated uptake influences the presentation of a protein quaternary structure. Unprimed splenocytes and a B cell lymphoma were capable of presenting only the free (hCG-α) but not the combined (hCG) α subunit to hCG-α T cell hybridomas, while hCG-α-primed lymph node cells (LNC) responded to both hCG-α and hCG. As antigen (Ag)-specific antigen-presenting cells (APC) present in the hCG-α-primed LNC population may be potentially effective for presenting hCG, we investigated the role of specific Ag capture, through mIg and FcγR, in the processing and presentation of hCG and hCG-a to HAG 5, a T cell hybridoma directed against the immunodominant region (amino acids 61-81) of hCG-α. Results showed that only B cells bearing membrane immunoglobulin capable of recognizing hCG-α and hCG, and present in hCG-α-primed mice, were extremely effective in presenting the free as well as the combined a subunit. The effect of FcR-mediated uptake was analyzed using a B cell line transfected with the FcγRII-B2 gene to present immune complexes of either hCG-α or hCG. We found that hCG-α and hCG were presented equally well, whatever the Ag-binding site of each antibody to hCG or its a subunit. Using HBG 6, an hCG-β Tcell hybridoma, we performed similar experiments with the FcγRII-B2 cell line and determined that the potentiation of hCG presentation to HBG 6 was similar to that observed with HAG 5. Then kinetic experiments were performed to examine the effect of Ag uptake through FcR on processing. Results demonstrated that the uptake pathway drastically influenced the expression of α T cell determinants in the αβ dimer. In addition, treatment with cycloheximide, a protein synthesis inhibitor, only impaired the ability of APC to present specifically captured Ag. Thus, the processing pathway for specifically captured Ag might be different from the pathway used to process nonspecifically captured Ag. This observation might explain why receptor-enhanced uptake bypasses the inefficient processing of the hCG quaternary structure and enables similar efficiency in the presentation of α and β T cell specificities. These findings provide new insight into the antigenicity of oligomeric molecules, which is modified whether antigen capture is specific or not.     

Toll-like receptor stimulation as a third signal required for activation of human naive B cells

According to the current model, naive B cell activation is dependent on the sequential integration of two signals: B cell receptor (BCR) cross-linking by antigen, followed by cognate interaction with helper T cells through an immunological synapse. Using an improved method to purify human naive B cells we found that BCR stimulation and T cell help induced initial cell division but were not sufficient to promote survival and differentiation thus leading to abortive proliferation of naive B cells. Extensive B cell proliferation, isotypic switch and differentiation to immunoglobulin (Ig)-secreting cells was induced by addition of microbial products that trigger any of the Toll-like receptors (TLR) that are up-regulated in naive B cells upon BCR triggering. TLR agonists acted directly on B cells and were required irrespective of the nature of the T helper cells present. Supernatants of dendritic cells (DC) stimulated by DC-specific TLR agonists were also capable of enhancing B cell responses although to a much lower and variable extent. These results indicate that human naive B cell activation is critically dependent on innate stimuli acting optimally on TLR expressed by B cells. The coupling of BCR stimulation to TLR expression endows the human system with a high degree of specificity since it allows focusing of innate signals only on antigen-stimulated B cells.     

A role for mitochondria in antigen processing and presentation

Immune synapse formation is critical for T-lymphocyte activation, and mitochondria have a role in this process, by localizing close to the immune synapse, regulating intracellular calcium concentration, and providing locally required ATP. The interaction between antigen-presenting cells (APCs) and T lymphocytes is a two-way signalling process. However, the role of mitochondria in APCs during this process remains unknown. For APCs to be able to activate T lymphocytes, they must first engage in an antigen-uptake, -processing and -presentation process. Here we show that hen egg white lysozyme (HEL) -loaded B lymphocytes, as a type of APC, undergo a small but significant mitochondrial depolarization by 1–2 hr following antigen exposure, suggesting an increase in their metabolic demands. Inhibition of ATP synthase (oligomycin) or mitochondrial Ca²⁺ uniporter (MCU) (Ruthenium red) had no effect on antigen uptake. Therefore, antigen processing and antigen presentation were further analysed. Oligomycin treatment reduced the amount of specific MHC–peptide complexes but not total MHC II on the cell membrane of B lymphocytes, which correlated with a decrease in antigen presentation. However, oligomycin also reduced antigen presentation by B lymphocytes, which endogenously express HEL and by B lymphocytes loaded with the HEL_48–62 peptide, although to a lesser extent. ATP synthase inhibition and MCU inhibition had a clear inhibitory effect on antigen processing (DQ-OVA). Taken together these results suggest that ATP synthase and MCU are relevant for antigen processing and presentation. Finally, APC mitochondria were found to re-organize towards the APC–T immune synapse.     

Differential presentation of hepatitis B S-preS(2) particles and peptides by macrophages and B-cell like antigen-presenting cells.

Different cell types, including dendritic cells, macrophages and Ia+ B cells, have been described to present soluble antigen (Ag) to T-cell hybridomas. However, it is still not clear whether these different cell types can act as antigen-presenting cells (APC) for complex and insoluble Ag such as viral particles. Using yeast recombinant hepatitis B S-preS(2)-containing particles, T-cell hybridomas were generated and used as a tool to study processing and presentation of antigen. Different types of APC were compared in regard to their capacity to process and present the protein-lipid composed S-preS(2) particles and the thereof derived T-cell epitope containing peptides by different types of APC. While a S-preS(2)-derived T-cell epitope containing peptide, which does not require processing, could be presented both by macrophage and B-cell like APC, the presentation of S-preS(2) particles required the presence of macrophages. The fact that B-cell like APC and macrophages behave differently with regard to the presentation of S-preS(2) particles suggest that the uptake and/or processing of this type of Ag by B-cell like APC and macrophages is different.     

B cell toll-like receptors with respect to the pathogenesis of Sjögren's syndrome

Sj?gren's syndrome (SS) is a chronic autoimmune immunopathological disease of unknown aetiology. It is characterized by focal lymphocyte infiltration and inflammation in exocrinne glands, involving especially salivary and lacrimal glands. Hypofunction of the glands leads to the decreased glandular secretion together with impaired production of saliva and tears, resulting in dryness of the mouth and eyes (xerostomia and xerophthalmia, respectively). Some of the studies have suggested that Toll-like receptors and B cells play a pivotal role in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis and SS etc. Stimulation of B cells via the TLRs pathway leads to several important changes including increase in antibody production, differentiation to plasma cells, cytokine production and up-regulation of molecules essential for antigen presentation to (autoreactive) T cells. Experimental data support the idea that co-engagement of BCR and TLR might be sufficient for B cell activation and lead to the failure of tolerance. In human naive B cells, most TLRs are expressed at very low or undetectable level, but expression of TLR 7 and 9 is rapidly induced by B cell receptor triggering. This review will focus on the possible role of B cells and TLRs signaling in the pathogenesis of SS.